To estimate the proficiency of inorganic pyrophosphatase as a catalyst, (31)P NMR was used to determine rate constants and thermodynamics of activation for the spontaneous hydrolysis of inorganic pyrophosphate (PP(i)) in the presence and absence of Mg(2+) at elevated temperatures. These values were compared with rate constants and activation parameters determined for the reaction catalyzed by Escherichia coli inorganic pyrophosphatase using isothermal titration calorimetry. At 25 °C and pH 8.5, the hydrolysis of MgPP(i)(2-) proceeds with a rate constant of 2.8 × 10(-10) s(-1), whereas E. coli pyrophosphatase was found to have a turnover number of 570 s(-1) under the same conditions. The resulting rate enhancement (2 × 10(12)-fold) is achieved entirely by reducing the enthalpy of activation (ΔΔH(‡) = -16.6 kcal/mol). The presence of Mg(2+) ions or the transfer of the substrate from bulk water to dimethyl sulfoxide was found to increase the rate of pyrophosphate hydrolysis by as much as ∼ 10(6)-fold. Transfer to dimethyl sulfoxide accelerated PP(i) hydrolysis by reducing the enthalpy of activation. Mg(2+) increased the rate of PP(i) hydrolysis by both increasing the entropy of activation and reducing the enthalpy of activation.